Dec 09, 2025
DNA Bisulfite conversion, using Zymo EZ-96 DNA Methylation-Lightning MagPrep Kit and Integra VIAFLO 96, for processing on Illumina methylation arrays
- Emma Aitken1,
- Nicola Wrobel1,
- Lee Murphy1
- 1Genetics Core, Edinburgh Clinical Research Facility, University of Edinburgh
Protocol Citation: Emma Aitken, Nicola Wrobel, Lee Murphy 2025. DNA Bisulfite conversion, using Zymo EZ-96 DNA Methylation-Lightning MagPrep Kit and Integra VIAFLO 96, for processing on Illumina methylation arrays. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbkj9qgpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 17, 2025
Last Modified: December 09, 2025
Protocol Integer ID: 220381
Keywords: dna methylation analysis service, throughput dna bisulfite conversion method, illumina methylation arrays the genetics core, dna bisulfite conversion, illumina methylation array, dna methylation, analysing dna sample, integra viaflo 96 platform
Abstract
The Genetics Core of the Edinburgh Clinical Research Facility provides DNA methylation analysis services using optimized automated protocols. This protocol details a high-throughput DNA bisulfite conversion method that uses Zymo EZ-96 DNA Methylation-Lightning MagPrep Kits and automated using the Integra VIAFLO 96 platform. The method is used as the first step in analysing DNA samples on Illumina Infinium Methylation Screening Arrays-48. This protocol allows 192 samples to be processed together.
Guidelines
This protocol details a high-throughput DNA bisulfite conversion method that uses Zymo EZ-96 DNA Methylation-Lightning MagPrep Kits and automated using the Integra VIAFLO 96 platform. The method is used as the first step in analysing DNA samples on Illumina Methylation Screening Arrays.
This protocol allows 2x 96-well plates (192 samples) to be processed together.
Materials
Zymo Research EZ-96 DNA Methylation-Lightning MagPrep Kit (D5046)
Integra VIAFLO 96 electronic pipette
Integra VIAFLO 96 Racks Sterile 300 µl (6434)
Integra VIAFLO 96 Racks Sterile Filter 300 µl (6435)
ThermoMixer e.g. Eppendorf ThermoMixer C (2231001127)
PCR Thermocycler e.g. Eppendorf Mastercycler X50 (2231001152)
Zymo Research ZR-96 Magnetic Stand (P1005)
Troubleshooting
Safety warnings
Step 19 is crucial. Samples need to be incubated for at least 15 mins and no longer than 25 mins. This limits the throughput of plates per operator.
Buffer Preparation
Add 288 mL of 95–100% ethanol to the 72 mL M-Wash Buffer concentrate. Mix well.
Label the buffer with preparation date and initials.
Sample Conversion
Transfer 20 µL of 50ng/ul (500ng) DNA into each well of the Conversion Plate.
Add 130 µL of Conversion Reagent (pre-mixed and light-sensitive). Mix thoroughly.
Seal with foil seal, centrifuge at 280 g for 30 seconds.
Place plate into the Eppendorf Mastercycler with settings:
98°C for 8 minutes
54°C for 60 minutes
Hold at 4°C (up to 20 hours)
Preparation of Collection Plate During Incubation
Add 600 µL of M-Binding Buffer and 10 µL of EZ-Methylation MagPrep Beads to each well of a fresh Collection Plate.
Keep beads suspended by gently vortexing or pipetting as needed.
Transfer to Collection Plate
Spin conversion plate at 280g for 30 seconds
Using VIAFLO 96 mix programme (aspirate 170ul at Asp speed = 1 and Mix speed = 8) and 300 µL sterile filter tips, transfer entire sample volume from Conversion Plate to corresponding wells of Collection Plate.
In collection plate, use VIAFLO 96’s mix function: mix 300 µL × 6 cycles to ensure bead binding. Dispose of tips.
Incubate at room temperature for 5 minutes.
Bead Separation
Place Collection Plate onto the ZR-96 Magnetic Stand for 5 minutes to pellet the beads.
Remove supernatant carefully using VIAFLO 96 at lowest speed setting. (Aspirate 300 µL at Asp speed = 1 and Disp. speed = 3) using 300 µL sterile tips (these do not need to be filter tips). Dispose tips
Wash Step
Remove plate from magnetic stand.
Add 400 µL of M-Wash Buffer, mix for 30 seconds at 1100 rpm on Eppendorf ThermoMixer C.
Return plate to magnetic stand for 3 minutes, remove supernatant.
Desulphonation
Add 200 µL of L-Desulphonation Buffer, mix on ThermoMixer for 30 seconds at 1100 rpm.
Incubate at room temperature for 15 minutes.
Place on magnetic stand for 3 minutes, remove supernatant.
Note: Take time for handling/re-suspension into account for the total incubation time. Adjust time as necessary to ensure that no sample remains in the L-Desulphonation Buffer for more than 20–25 minutes. This is particularly important when processing two plates together.
Final Washes and Elution
Add 400 µL of M-Wash Buffer, mix for 30 seconds at 1100 rpm, return to magnetic stand for 3 minutes, and discard supernatant.
Repeat the wash step once more (total: 2 washes).
Note: Remove as much buffer as possible after final wash to aid in the drying of the beads.
After second wash, dry beads by heating the plate at 55°C for 25 minutes on the ThermoMixer.
DNA Elution
Add 25 µL of Elution Buffer. Mix for 30 seconds at 1300 rpm on the ThermoMixer.
Heat the elution at 55ºC for 4 minutes then transfer the plate to the magnetic stand for 1 minute. Remove the supernatant and transfer to a clean Elution Plate using VIAFLO 96 pipette function (Aspirate 30ul at Asp speed = 1 and Disp. speed = 3) and 300 µL sterile filter tips.
Eluted DNA is now ready for downstream analysis or can be stored at -20°C.
Protocol references
Zymo EZ-96 DNA Methylation-Lightning MagPrep Kit Protocol Ver.2.1.1
Accessed 1 Dec 2025
Illumina Infinium EX Methylation Assay Semi-Automated Workflow
Document #200048699 v01
Accessed 1 Dec 2025
Integra VIAFLO 96 Instruction Manual 125950_V21
Accessed 1 Dec 2025